Combinatorial Synthesis and Discovery of an Antibiotic Compound Developed by Scott E. Wolkenberg and Andrew I. Su, The Scripps Research Institute

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1 Introduction and Background: Combinatorial Synthesis and Discovery of an Antibiotic Compound Developed by Scott E. Wolkenberg and Andrew I. Su, The Scripps Research Institute Biology and chemistry interact in many ways. The two fields affect one another greatly, and experts in each field often work in the other. The reason for this interaction and influence is the success each has had in increasing our understanding of the other. For instance, all of the components of living organisms are molecules, and these in turn are made up of atoms. The dizzyingly complex interactions of these many molecules lead to the functions and behaviors of living systems. While the study of living systems is surely biology, the study of interacting atoms and molecules is undoubtedly chemistry. As a result, chemists have been drawn to the study of these wonderfully complicated chemical systems, and they have made discoveries that have advanced our understanding of biological systems tremendously. Biology has had a similar impact on chemistry. Chemistry is chiefly concerned with reactions, with transforming one substance into another. Despite great advances in chemical understanding of reactions, there remain reactions that chemists cannot perform under reasonable conditions. There exist, however, biological systems that perform such reactions routinely under mild conditions. By studying these systems, biologists and chemists have learned of reaction pathways and molecular interactions they never could have anticipated. ne of the areas in which there is the most interaction between chemistry and biology is drug discovery. The very nature of a drug requires consideration of both chemistry and biology since it is, by definition, a chemical interacting with a biological system to effect some change. The way drugs are discovered has changed dramatically in the past years due to the development of a new methodology for synthesis called combinatorial chemistry. In this experiment, we aim to expose you to this method and express its usefulness in drug discovery. Combinatorial chemistry is a method for discovering molecules that possess a desired property. The property can be almost anything: electrical conductivity or semiconductivity, an attractive color, a sweet odor, the ability to block pollen from binding to a histamine receptor, the ability to kill cancerous tumor cells, the ability to kill bacterial cells. What distinguishes combinatorial chemistry is its ability to screen many (thousands to millions) of new compounds for a desired property. istorically, the way compounds with desired properties were discovered and developed was by finding some "lead compound" in nature which exhibited the desired property. The chemical structure of lead compounds were then modified using chemical reactions in order to optimize the property. This approach has proven effective (it is responsible for most of the drugs currently available), but it is very slow since each compound is synthesized individually. Combinatorial chemistry changes the paradigm for discovery by introducing the notion that compounds should be synthesized as mixtures. When mixtures are produced in chemical reactions, the number of compounds produced increases exponentially. For example, consider a reaction which joins two types of molecules, A and B, to form a molecule A B. If, as has historically been done, one molecule of each component is combined in a reaction, one product molecule results: A1 + B1 A1 B1. If, however, two of each component is included in the same reaction vessel, four product compounds are produced: A1 + A2 + B1 + B2 A1 B1 + + A2 B1 + A2 B2. If three of each component is included in the same reaction vessel, nine product compounds are produced, and so on. By combining more and more components in these types of reactions, huge mixtures (or "libraries") can be produced. The number of tests for the desired property is greatly reduced if those tests are carried out on mixtures of compounds instead of individual compounds. (It is much simpler to perform an antibiotic screen on one mixture of 1000 than on 1000 individual compounds.) nce it has been determined that a mixture has the desired property, the problem changes to identifying which of the many compounds in the mixture is the active one. The process of making that determination is called "deconvolution." There are many methods for deconvoluting an active mixture of compounds, each with advantages and disadvantages. Regardless of the specific method employed, it is possible to identify active compounds performing fewer chemical reactions and fewer tests for the desired property than if the compounds were synthesized and tested individually. Identification of an antibiotic using combinatorial chemistry: In this experiment, you will produce libraries of compounds based on the A B model discussed above. You will simultaneously generate 6 libraries, test them for antibiotic activity, and deconvolute them to discover which individual compound has antibiotic properties. S1

2 The chemical reaction which forms the basis for this reaction is the condensation of an aldehyde (A) and a hydrazine (B) to form a hydrazone (A B) and water. R 1 C 2 R 2 R 2 R 1 C A B A B The parts of the molecules labeled with "R" and numbers represent the variable parts. They can be almost any grouping of atoms, so long as they do not interfere with the reaction shown above. A shorthand for describing the reaction above is to say A1 + B2. In this experiment, three aldehydes (A) and three hydrazines (B) will be combined in a combinatorial procedure to produce 9 hydrazones (A B). The structures of the six components are shown below. Br 2 A1 A2 A3 C C C B1 B2 2 C 2 C 2 2 You will then screen the mixtures for antibiotic activity. This method entails the growth of a "lawn" (a uniform layer) of bacterial cells on a solid support (agar). You will then lay paper disks treated with a library of compounds on top of the agar. The solutions will diffuse through the agar about 2 cm from the disk. If the solutions contain a compound with antibiotic activity, there will be no growth in a circle surrounding the disk. Laboratory Safety:* The bacterial host used in most molecular biology and teaching laboratories is Escherichia coli. Since E. coli is often associated with outbreaks of disease, concern may arise over its safety. Unfortunately, media reports on E. coli disease do not contain the background information necessary for understanding this issue. There are many naturally occurring strains of E. coli. They inhabit the lower intestinal tracts of many animals, including humans, cattle, and swine. The strains found in different animals vary genetically, and can even vary between individuals of the same species. owever, some genetic variants of E. coli do cause disease. These variants contain genes not found in the harmless organisms. These genes encode toxins and proteins that enable the organism to invade cells within the body. The nature of the disease gene varies; E. coli strains with different disease genes have been associated with several diseases. Some E. coli have genes for enterotoxin, which causes the travelers' diarrhea. The E. coli that causes the sometimes fatal hemolyticuremic syndrome have genes that encode a toxin different from the travelers' diarrhea toxin, and additionally have genes that enable them to invade and disrupt cells lining in the intestinal tract. S2

3 Laboratory strains of E. coli used in molecular biology research do not contain any of these disease genes and are harmless under normal conditions. If introduced into a cut or into the eye, laboratory strains could conceivably cause infection, so standard safety precautions should be taken when handling the organisms. Every day, hundreds of scientists and their students handle these organisms (many in a rather cavalier manner) without any notable consequences. We do not recommend cavalier handling of any strain of E. coli, but the history of scientists with the organism should be reassuring. Tips for andling E. coli:* Wipe down lab bench with a 10% bleach solution, soapy water, or disinfectant at the end of laboratory sessions. Wash hands before leaving laboratory. Collect for treatment bacterial cultures, as well as tubes, pipets, and any other materials that have come into contact with cultures. Disinfect these materials in one of two ways: o Treat with 10% bleach solution for 15 min or more before disposing in the regular garbage. o Autoclave at 121 C for 15 min. Dispose of sterilized materials in the regular garbage. *adapted from the Teacher's Manual accompanying # Petrifilm Transformation Kit, Carolina Biological Supply Co. Chemical safety is a serious concern and great care has been taken to ensure that the chemicals used in this experiment pose as little health threat as possible. The chemicals included in this experiment are not toxic in the amounts used. For instance, compound A1, 2-nitrobenzaldehyde, is toxic to a typical adult when 18 g (18,000 mg) are consumed orally. In this lab, 54 mg of this compound will be used for the entire class. Despite the lack of toxicity, some of the compounds are irritating to the skin and eyes so standard laboratory safety procedures should be followed. ands should be washed when leaving the laboratory, and any spills should be reported and cleaned up immediately. Lab Procedure: TE: KEEP PETRI DISES CVERED AT ALL TIMES. REDUCE YUR RISK F CTAMIATI BY UCVERIG TEM LY WE USIG TEM. 1. Using a marker, label the bottoms of two petri dishes (previously inoculated with a bacterial culture) as shown below. The bottom is the side which contains the yellow agar. Do not remove the top cover of the plates. Divide each of the petri dishes into 3 sections. Label the sections of one plate M1, M2, M3, and the sections of the second plate,,. In addition, label the plates with your initials to distinguish them from your classmates. Turn the plates back over so the bottom is down and the top is up. M1 M2 M3 SEW SEW 2. Using the transfer pipet for each solution, add the appropriate reagents to each of the six 1.5 ml tubes according to the table below. Be sure to add the reagents in the proper order and be sure to add the correct number of drops. For instance, to prepare M1, add 5 drops B1 to tube M1, then add 5 drops B2 to tube M1, then add 5 drops to tube M1, then add 15 drops A1 to tube M1. Tube # Add 5 drops then 5 drops then 5 drops then 15 drops M1 B1 B2 A1 M2 B1 B2 A2 M3 B1 B2 A3 A1 A2 A3 B1 A1 A2 A3 B2 A1 A2 A3 Record any observations you make during the addition. S3

4 3. Cap each tube and shake for seconds. Again record your observations of the mixtures. 4. Using a pair of forceps washed with ethanol, dip a paper disk briefly in the contents of tube M1 Allow any excess to drip back into the tube, and then gently place the paper disk into the center of the petri dish region labeled M1. Rinse the forceps with ethanol, and repeat for the remaining 5 mixtures. 5. Incubate the two agar plates at 37 C for 24 h. Be careful when carrying the plates to the incubator. Data Analysis: Please listen in lab for details on viewing your incubated plates 1. Using the table below, record the results of the experiment. Place a "+" in the table for mixtures that resulted in an inhibition of growth. Place a " " in the table for mixtures that resulted in no inhibition of growth. Include a copy of this table in your lab notebook/report. Mixture Contents Result M1 A1, B1, B2, M2 A2, B1, B2, M3 A3, B1, B2, B1, A1, A2, A3 B2, A1, A2, A3, A1, A2, A3 Each mixture contains three compounds. For instance, M1 contains the compounds A1 B1, A1 B2, and. Therefore, in the mixtures that show inhibition of growth (antibiotic activity) there are three compounds. ow can we tell which of the three compounds is the one with the antibiotic activity? In fact, we have already done the experiments needed to make this determination. 2. The table below enables us to determine all the compounds we made. The outside of the table shows the starting materials for the reactions we performed: A1, A2, A3, B1, B2, and. Whenever an A molecule comes in contact with a B molecule they react to form A B molecules. We can see this in the table by looking at the inside: when we trace A1 down in the first column, we see that it forms A1 B1, A1 B2, and molecules. Fill in the rest of the table in the same way by tracing across and down. A1 A2 A3 B1 B2 3. The inside of the table represents the contents of each mixture M1. For instance, the mixture M1 is represented by the first column in the table (see the shading in the left table). Likewise, M2 and M3 are represented by the second and third columns. Mixture is represented by the first row in the table (see the shading in the right table). Similarly, and are represented by the second and third rows of the table. 4. We can use this table to determine which compound in our active mixtures shows antibiotic activity. Fill in the table below as you did in part 2. Shade in the column that corresponds to the mixture M1 M3 that shows antibiotic S4

5 activity. ow shade in the row that corresponds to the mixture that shows antibiotic activity. The position in the table where the shaded row and shaded column intersect is the active compound. Determine the best antibiotic compound made in this experiment, and clearly report that in your discussion/conclusion. Post-lab questions: 1. What is the advantage to working with mixtures? ow many reactions would we need to run in order to make each compound individually? ow many antibiotic activity tests would we need to run in order to screen each compound individually? Compare these numbers with the number of reactions and antibiotic activity screens we ran in this experiment. 2. What might be some possible disadvantages to working with mixtures? 3. Consider a larger system, one with 10 hydrazines and 10 aldehydes. A. ow many possible hydrazones can be made from these starting materials? B. Using the same methods described in this lab, how many chemical reactions and antibiotic screens are necessary to identify any active hydrazones? C. Plot the number of chemical reactions and antibiotic screens as a function of the number of compounds synthesized and screened for antibiotic activity for a) the traditional (one at a time) method and b) the combinatorial method described in this lab. D. Evaluate the method of deconvolution used in this lab using the example data shown below for a large (10 10) system. Can you imagine any other circumstances in which, aside from synthesizing individual compounds, there is no way to determine which compound is active? M7 M8 M9 M10 M11 1M12 M13 M14 M15 M16 M17 M18 M19 M20 B1 B2 B4 B5 B6 B7 B8 B9 B10 A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 4. Can you imagine any other deconvolution strategies? Remember the goal is to minimize the number of chemical reactions and antibiotic screens required to unambiguously identify active compounds. 5. Propose a mechanism for the reaction that formed any active antibiotic in your experiment. S5

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